Short inverted repeated (palindromic) sequences are present at several functionally important regions in both prokaryotic and eukaryotic genomes. However, longer inverted repeats (greater than 150 base pairs) are less frequently observed and are difficult to maintain in Escherichia coli. The mechanisms underlying the preservation of short inverted repeats in the genome and elimination of longer sequences from the genome are not clearly understood. Palindromic sequences have the potential to form hairpin or cruciform structures, which are putative substrates for several nucleases and mismatch repair enzymes. The central hypothesis to be tested in this proposal is that the instability of long palindromic sequences is caused by cruciform formation which leads to processing by structure-specific nucleases. The long-range goal of this research program is to enhance our understanding of the mechanism of genomic instability caused by the presence of palindromic sequences. The unicellular eukaryote Saccharomyces cerevisiae will be used as a model eukaryote for these studies. The specific aims address: 1) the mechanism of the meiotic double-strand break (an initiator of meiotic recombination) formation by a long palindromic insertion-mutant allele, 2) the identification of the gene(s) responsible for making the double-strand break (DSB), and 3) the identification of proteins that interact with the enzyme that makes the DSB. The experimental approach utilizes different genetic and physical methods to establish the pathway for DSB formation, to map the DSB sites at the nucleotide level, to determine the role of the primary sequence of the inverted repeat, and to determine the length of the inverted repeat required to function as a hotspot for meiotic recombination. A genetic screen will be used to identify the gene responsible for making the DSB, and the interacting proteins will be identified by two-hybrid and high-copy suppressor analysis. It is anticipated that these studies will uncover the mechanisms of palindrome-mediated genomic instability, and an important step of the genetic recombination process. Recombination plays an important role in meiotic chromosome segregation, and DNA lesions often result in chromosomal translocations, a common cause of many leukemias and lymphomas. Thus, the results of these studies will contribute significantly to our understanding of some forms of malignancy as well as several reproductive diseases.